Birth of babies with congenital defects
is the most traumatic experience to the expectant parents.
The incidence of neural tube defects and chromosomal
aneuploidy are quite high. Since a positive correlation was
found with maternal age, early workers offered the choice of
amniocentesis to detect a defective fetus. Amniocentesis
posed a risk for the fetus which was as high as the chance
of having an affected child, several groups worked on the
development of non invasive prenatal screening tests.

All three types of aneuploidy have a strong correlation with
maternal age above 35 years. Since the changing lifestyle
has increased the maternal age above 35, reliable markers
with high detection rate and low false positive are found to
be essential.

Thus several biochemical markers were measured in maternal
serum and have now been identified as reliable indices of
neural tube defects and chromosomal aneuploidy. Most of
these markers are normally present in maternal serum and
their level varies with gestational age. Hence these markers
are said to have temporality. So both the patient and
requesting physician must have a clear idea that the result
of a screening test is different from that of a diagnostic
test. In order to confirm the diagnosis of a chromosomal
disorder, invasive procedures like Chorionic villous
sampling or amniocentesis should be done and cytogenetic
studies on the cells to be carried out.

Unlike markers used in other diseases, the levels of
biochemical markers used in prenatal diagnosis are expressed
by the statistical term Multiple of Median (MOM). The
markers are quantitated by any of the modern techniques like
RIA/EIA/CLIA and then converted to MOM. MOM values normalize
results for purposes of comparison between laboratories
.Each laboratory has to use its own data to establish Median
values for each marker (analyte) for each week (day) of
gestation. Ideally the median is to be calculated with
results from 200 samples. MOM values are obtained by
dividing the actual value with the lab Median. Kit specific
and population specific median values increase reliability
and the MOM should be updated.

Since the 1990’s prenatal screening has become standard
obstetric practice in all pregnancies at risk. Since
maternal age is associated with errors in meiosis an
increase in maternal age increased the risk of having
children with chromosomal aneuploidy. This is one of the
primary indications for prenatal screening.

The Triple Test (Triple screen)

The triple screen is done during the second trimester
between 14 and 18 weeks. The markers used are AFP, uE3 and
HCG. The pattern of changes and detection rates for Neural
tube defects, Trisomy 21 and trisomy 18 are given in table.

Alpha fetoprotein is the major serum protein
of the fetus synthesized by the fetal liver and yolk sac.
There is a steady increase in AFP level in maternal serum
from 10th week of gestation and reaches a peak by 25 weeks
of gestation in unaffected pregnancy. Then the maternal
serum alpha feto protein (MSAFP) steadily declines until
term. In fetal serum and amniotic fluid, the AFP level
reaches a peak by 9th week of gestation and then slowly
falls till term. In NTD, the AFP is increased but in
chromosomal aneuploidy it is decreased.

Human Chorionic Gonadotrophin is a
glycoprotein hormone produced during normal pregnancy by the
trophoblast and placenta. HCG appears in maternal serum by 6
to 8 weeks and reaches a peak by 10 weeks. By the second
trimester it falls to a constant level by 18 to 20 weeks.

A marked increase about twice the normal was found in
pregnancies with trisomy 21 during the second trimester. It
was also noted that free beta HCG was increased during the
1st trimester in DS (MOM 2.4) even though total HCG remained
normal (MOM 1.33). Both were increased during the second
trimester in Trisomy 21(MOM 2 to 2.5). A hyperglycosylated
variant (produced by cytotrophoblasts) was also found in
Down’s syndrome. This is referred to as Invasive Trophoblast
Antigen (ITA). The higher level of ITA is believed to be due
to the defect in the conversion of cytotrophoblast to
syncytiotrophoblasts. In trisomy18 the HCG levels remained
lower than normal.

Unconjugated Estriol (uE3) is a steroid
hormone produced by the fetoplacental unit .It is produced
by the placenta but the fetal liver completes the synthesis.
It is an estrogen with 3 hydroxyl groups and 3 organs –fetal
adrenal, fetal liver and maternal liver are involved in the
synthesis. Initially estriol was used as a measure of fetal
wellbeing especially at term. Maternal serum uE3 levels rise
by 8weeks of gestation and continue to increase through out
pregnancy. A 25 % reduction in uE3 levels was found in
maternal serum when the fetus had chromosomal aneuploidy.

Even though the Triple screen has a high detection rate,
temporalities of the markers have to be emphasized. During
the second trimester, AFP and uE3 increase in unaffected
pregnancies, where as that of HCG declines. Yet another
point which can give false positive results is the method of
calculation of gestational age- by ultrasound or by LMP.
This fallacy can be corrected using the USG results at 6
weeks of gestation for calculating correct gestational age.

In spite of these limitations, the triple screen has a high
detection rate, 80% for neural tube defects and 55-60% for
chromosomal aneuploidy and a false positive less than 5 %.
When the cut off is around 1:270,false positive rises to 7
to 9%.The increase in maternal age with the changing
lifestyle, career options and work pressure in educated and
employed women have increased the need for additional
markers.

The Quadruple Test (Quad screen)

Includes AFP.uE3, HCG and an additional marker Inhibin-A

Dimeric Inhibin A (DIA) is a glycoprotein
produced by the placenta. It is a Dimer, but with dissimilar
subunits alpha and beta. Inhibin A has the subunit make up
ab-A and inhibin B a-b B .Inhibin A is measurable in
maternal serum and has a feed back effect on FSH secretion.

The level increases in the first trimester until 10 weeks
and then remains stable upto 25 weeks of gestation.
Thereafter it increases to reach a peak by term. The DIA
levels are increased in DS and remains elevated through out
the second trimester unlike AFP and uE3 that increase and
hCG that decreases during the testing period.

It is the least stable of the four analytes, since
decomposition can occur during storage. Serum should be
promptly separated and assay completed within 3 days. Whole
blood should not be transported. 0.7 –2.5 microgram/L in
unaffected pregnancy at second trimester.

Statistical modeling of different combinations of AFP, uE3,
hCG and DIA indicates that the highest detection rates and
lowest false positive rates are achieved when using all four
markers corrected for maternal weight. DIA is an independent
variable having no correlation with maternal age, race or
Insulin dependent diabetes mellitus. The only factor which
has a significant effect on DIA levels is maternal weight.
There was no correlation with AFP and uE3, but significant
correlation was found with hCG

Factors affecting the level of the Quad screen markers

Maternal Weight was found to have an inverse
relation with the levels of all four markers.

Diabetes- Insulin dependent (not
GDM) AFP was found to be 40% lower than non diabetics

Twin pregnancy- MSAFP higher than
those having single fetus. MOM > 4 significant

Racial differences were also noted,
but not significant in our set up

Screening during the First trimester

Several workers suggested that a screening test done
between 10 and 14 weeks of pregnancy (1st trimester) may
be as accurate as that done during the second trimester.
In this screening method, AFP, hCG and Pregnancy
associated plasma protein-A(PAPP-A) are measured along
with ultrasound examination for Nuchal Translucency(NT)
This screening pattern is referred to as an OSCAR(One
Stop Clinic for Assessment of Risk) .All the markers are
measured in a single serum sample.

PAPP-A is a high molecular weight zinc containing
metalloglycoprotein. It is produced by the trophoblast
and its biological function is still unclear. In
addition to being a marker of chromosomal aneuploidy, it
is an indicator of early pregnancy failure and
complications, Cornelia de Lange syndrome and acute
coronary syndrome.

The level of PAPP-A was found to be significantly lower
in pregnancy with Trisomy 21 compared to unaffected
pregnancy (MOM 0.27 as against 1.01 in normal
pregnancy)But PAPP-A levels when measured in second
trimester the results are found to be normal.
Persistently lower levels of PAPP-A in second trimester
was indicative of Trisomy18.

Similarly total hCG was found to be a poor marker in the
first trimester with an MOM of 1.33, but an adequate
marker during the second trimester. Free beta hCG on the
other hand is higher and has a relatively stable MOM (2)
from 10 to 18 weeks.

The detection rate was 89% with 5% false positive.

Maternal serum marker(10-14 weeks)

NTD

Down’s syndrome

Trisomy -18

Pattern

PAPP-A

Low

Low

MOM 0.45

Total beta hCG

Normal

MoM-1.33

Free beta hCG

Increased

MOM 2.0

Hence the present suggestion is to combine the markers
of first and second trimester in maternal serum. The
suggested protocol is given

Measurements of NT and PAPP-A are made in the first
trimester, but not interpreted or acted upon until the
second trimester.

In the second trimester a second serum sample is drawn
and quadruple test performed.

Results for all the six tests, NT, PAPP-A, AFP, uE3,
hCG and DIA are combined into a single risk estimate for
interpretation in the second trimester.

85% detection rate for DS with only 1% false positive
is achieved

Information to be provided by Physician

1. Specimen collection date2. 1st or second specimen3. date by LMP or gestational age by USG4. maternal age and weight5. relevant family and obstetric history6. presence of maternal Diabetes mellitus7. Maternal race if indicated
8. Whether multiple pregnancy is present/suspected

ReportingThe report should contain

1. MOM values for the measured analyte2. DS risk estimate along with risk for NTD or trisomy
183. an interpretation as screen positive or screen
negative4. information that suggests possible further action.

Interpretation of screen results and calculation of risk

It is seen that about 5% test screen positive, for DS,
3% for NTD and 0.2 % for Trisomy 18, but inaccurate
dating can give an abnormal screening result. Ultrasound
dating gives the best dating accuracy. The MOM values
are adjusted according to the patient’s gestational age,
maternal weight, insulin dependent diabetic status and
twin pregnancy.

The AFP, hCG, uE3 and DIA values are then compared to
known data for affected and unaffected pregnancies and a
likelihood ratio is calculated for each marker. The
likelihood ratios are then combined with the patient’s
age related risk for DS. The resulting number is the
patient’s specific risk for DS. There are no reference
intervals for each analyte.

The detection rate and false positive rate are
determined by the screen positive cut off level.
Maternal serum marker levels are used to modify a
pregnant woman’s age related risk (priori risk) to
calculate the patient specific risk. A test is said to
be positive if a woman’s fetal DS risk by maternal serum
screening is the same as or greater than the fetal DS
risk of a 35 year old woman. At 35 years, the risk
during second trimester is 1/270 (with 1/350 at term).
Using a risk of 1/270 as a cut off level, four marker
screening will achieve a detection rate of 75 to 80%
which is 15 to 20% above the DR of Triple screen with
same false positive rate of 5%. However if the cut off
is fixed at 1/150 then the false positive falls to 3%.
This would ensure less patient anxiety and fewer
amniocenteses.

False positive rate signifies the proportion of women
with test results falling at or above a specified MOM
for AFP .But since several women are True positive, at
present the term Initial positive rate (IPR) is used
instead of FPR. The IPR can be used to assess whether
Medians are appropriate since it will be shifted upward
or downward if medians are incorrect.

Follow up of Patients with Screen positive results:

Genetic counseling if patient is screen positive

For moderately elevated results (MOM 2-3 ) a second
test

If second test is negative – Screen negative

If second test also gives elevated results, further
testing

USG, Amniocentesis and analysis of AF for Acetyl Choline esterase to confirm NTD

Amniotic fluid AFP results may give false positive due
to contamination by fetal blood, hence confirmed by
Acetyl choline esterase .Ach-E is not normally present
in AF, but appears in open neural tube defects since
fetal CSF contains the enzyme